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Abstract

A morphological review and molecular characterization of Anilocrahaemuli Bunkley Williams & Williams, 1981, were completed using specimens collected from Haemulonflavolineatum Desmarest, 1823 (French grunt) and Epinephelusguttatus Linnaeus, 1758 (red hind). Molecular and morphological data suggest that the isopods parasitizing H.flavolineatum and E.guttatus are different species. The specimens collected from E.guttatus are recognized as a new species, Anilocrabrillaesp. n. Differences between Anilocrabrillaesp. n. and A.haemuli include but are not limited to the pleonites 1–3 of A.brillaesp. n. being wider than 4–5 and 4–5 subequal, whereas the pleonites 1–2 of A.haemuli are wider than 3–5, and 3–5 are subequal. The seventh pereopod of A.brillaesp. n. is proportionally larger, has more robust setae, and the setae are distributed more extensively over the articles when compared to A.haemuli. Additionally, this study provides the first genetic characterization of three Anilocra spp. from the Caribbean, and is based on mitochondrial cytochrome c oxidase subunit gene (COI) for A.haemuli from H.flavolineatum, A.brillaesp. n. from E.guttatus, and A.chromis Bunkley Williams & Williams, 1981 from Chromismultilineata Guichenot, 1853.

Keywords

Introduction

In the past half-century, taxonomic studies on the fish parasitic isopod genus Anilocra Leach, 1818, have reported nine species from the Caribbean (Bunkley Williams and Williams 1981) and 12 species from Australia (Bruce 1987). This genus of parasite parasitizes the external surfaces of marine fish hosts that inhabit subtropical, tropical, and temperate waters (Smit et al. 2014). Host specificity of species of Anilocra is highly variable, such that different Caribbean Anilocra have been identified as family, genus, and species specific (i.e. Bunkley Williams and Williams 1981, Bruce 1987). For example, Anilocraholocentri Bunkley Williams & Williams, 1981 has been reported only to infest Holocentrusadscensionis Osbeck, 1765, whereas Anilocrachaetodontis Bunkley Williams & Williams, 1981 has been reported to infest four members of the genus Chaetodon Linnaeus, 1758. Anilocrahaemuli Bunkley Williams & Williams, 1981 is the only Caribbean species reported to infest fishes from two families: Haemulidae and Serranidae. Anecdotal accounts from both parasitologists and ecologists suggest that records of A.haemuli from Haemulids and Serranids may in fact be two species given the differences in the biology and ecology of these host fishes.

To evaluate this claim a review of Anilocrahaemuli morphology using specimens from both the Haemulidae and Serranidae families is warranted. The original description of A.haemuli was published before molecular approaches were used to aid in confirming the morphological classification of organisms. In the original description, careful attention was taken to describe A.haemuli as type specimens were collected from the same host and locality (Bunkley Williams and Willams 1981). Nevertheless, multiple morphologically similar species of Anilocra may have been identified as A.haemuli because there was no other method to verify if these specimens represented multiple species.

An increasing number of ecological studies are using Anilocra to understand trophic level dynamics (Roche et al. 2013, Binning et al. 2014), and A.haemuli infestation has been associated with altering H.flavolineatum behavior and condition (Welicky and Sikkel 2014, 2015, Welicky et al. in press). To facilitate future ecological and evolutionary studies on Anilocra–host interactions, the identity of Anilocrahaemuli is here validated using both a morphological redescription and a molecular analysis.

Materials and Methods

Specimen collection

In August 2016, Epinephelisguttatus Linnaeus, 1758, (family Serranidae) (n = 8) parasitized by a cymothoid isopod of the genus Anilocra were collected by free-divers using a modified cast net (Sikkel et al. 2004, 2006, Welicky et al. 2013) from Guana Island, British Virgin Islands (BVI). The Anilocra specimens were removed from host fish using forceps and then stored in 80% ethanol. Anilocrahaemuli from H.flavolineatum Desmarest, 1823, (family Haemulidae) (St. John, USVI, n = 4, 2011; n = 2, 2012; n = 1, 2013; Guana Island, BVI, n = 1, 2012; n = 2, 2013; St. Thomas, USVI, n = 2) were collected in a similar manner as part of other studies, and initially frozen and then preserved in 80% ethanol. To include a third and more morphologically distinct Anilocra sp., Anilocrachromis Bunkley Williams & Williams, 1981, infesting Chromismultilineata Guichenot, 1853 (St. John USVI, n = 8, 2012-2013) were also collected. These were collected in a similar manner to those of A.haemuli from H.flavolineatum.

Molecular analysis

Of the specimens collected, genomic DNA was extracted from eight Anilocra from E.guttatus, seven A.haemuli from H.flavolineatum, and eight A.chromis from C.multilineata using a rapid DNA extraction method as described in the KAPA Express Extract Kit (Kapa Biosystems, Cape Town, South Africa). Polymerase chain reactions (PCR) were used to amplify a 710 basepair fragment of the mitochondrial cytochrome c oxidase subunit gene (COI) using the primer sets LCO 1490 and HCO 2198 (Folmer et al. 1994). PCR was performed using 12.5 μl Thermo Scientific DreamTaq PCR master mix (2×) (2× DreamTaq buffer, 0.4 mM of each dNTP, and 4 mM MgCl2), 1.25 μl of each primer, 1 μl DNA, and 9 μl of PCR-grade nuclease free water (Thermo Scientific, Vilnius, Lithuania). Total volume per reaction was 25 μl, and PCR reactions were conducted using a ProFlex™ PCR thermal cycler (Applied Biosystems by Life Technologies). Reactions were amplified under the following PCR conditions: Stage 1, 94°C for 5min, Stage 2, 36 cycles of 94°C for 30s, 47°C for 50s, 72°C for 2min, and Stage 3, 72°C for 10min. PCR products were sent to a commercial sequencing company (Inqaba Biotechnical Industries (Pty) Ltd, Pretoria, South Africa) for purification and sequencing in both directions. Obtained sequences were assembled, and chromatogram-based contigs were generated using Geneious Ver. 9.1. Sequences were aligned and trimmed to the length of the shortest sequence using MEGA 7 bioinformatics software program (http://www.megasoftware.net)

Morphological data

Anilocrahaemuli from Haemulonflavolineatum and Anilocra from Epinephelusguttatus were examined using material previously collected by Ernest Williams and Lucy Bunkley-Williams during 1976–1977 and 1983 and reported in Bunkley-Williams and Williams (1981). Additionally, specimens from each host were collected using the aforementioned methods as part of other studies conducted in the US Virgin Islands (USVI) and British Virgin Islands (BVI) during 2011–2016. Isopods were processed according to the techniques described in Hadfield et al. (2010, 2013). Descriptions were prepared using DELTA (Descriptive Language for Taxonomy, Coleman et al. 2010) using a general character set for the Cymothoidae (Hadfield et al. 2014, 2016). Ratios and measurements were rounded off to one decimal place and were made using maximum values of the specific measured article. Ratios and measurements were taken from the female (♀) and transitional stage (TS) specimens used for the drawings and presented herein as figures. Pleotelson length (TL) and width (W) for all specimens examined are reported. All measurements are reported in milliimeters (mm). Classification follows Brandt and Poore (2003).

Results

Molecular analyses

Comparative sequence analysis indicated that there were three distinct species present in the samples based on the host species, A.haemuli from H.flavolineatum, A.chromis from C.multilineata and another undescribed species of Anilocra from E.guttatus. The intraspecific divergence observed within species was as follows: A.haemuli, 1–6 nt (0.6%); A. sp. n., 1–4 nt (0.3%); and A.chromis, 1–6 nt (0.7%) (Suppl. materials 1 and 2). The interspecific divergence between pairs of Anilocra spp. was as follows: A.haemuli and A. sp. n., 12–19 nt (4%); A.haemuli and A.chromis, 31–37 nt (9%); and A.chromis and A. sp. n., 31–37 nt (8%) (Suppl. materials 1 and 2). The interspecific divergence ranged between 104–109nt (30%) for all of our specimens combined and those available on GenBank (Suppl. materials 1 and 2).

Type species

Leach (1818) described three species: Anilocracuvieri, Anilocramediterranea Leach, 1818, and Anilocracapensis Leach, 1818 without designating a type species. A.cuvieri was designated as the type species by Kussakin (1979). Both Anilocracuvieri and A.mediterranea were synonymized with A.physodes (Trilles 1975; Ellis 1981).

Remarks

The body of female Anilocra is dorsally symmetrical and strongly vaulted. The posterior margins of their cephalon are smooth and straight, and the rostrum is more blunt than pointed. The rostrum folds into the area between the antennula bases. The antennula is shorter than the antenna. The posterolateral margins of the pereonites are not produced. Coxae 1–3 are short, posteriorly rounded and do not form a rounded point posteriorly, whereas coxae 4–6 are longer, less rounded and more elongate than coxae 1–3, and form a rounded point posteriorly. The pereopods gradually increase in size towards the posterior.

In the Cymothoidae, the external-attaching genera include but are not limited to Anilocra, Nerocila Leach, 1818, Renocila Miers, 1880, Creniola Bruce, 1987, and Pleopodias Richardson, 1910. Anilocra can be distinguished from Nerocila by the posterior margin of the cephalon, which is conspicuously trilobed in Nerocila, whereas the posterior margin of the cephalon of Anilocra is not tri-lobed to weakly tri-lobed. The posterolateral pereonite margins of Nerocila are more produced, elongate and pointed than that of Anilocra. In the Caribbean, some species of Anilocra and Renocila share numerous similarities, but in Anilocra pereopod 6 is shorter in length than pereopod 7, whereas in Renocila pereopods 6 and 7 are of similar length. To date the genera Creniola and Pleopodias have not been reported from the Caribbean.

Antennula consisting of 7–8 articles; peduncle articles 1 and 2 distinct and articulated; article 2 0.8 times as long as article 1; article 3 0.9 times as long as wide, 0.4 times as long as combined lengths of articles 1 and 2; flagellum with 5 articles, extending to posterior margin of eye. Terminal article with 2 short simple terminal setae. Antenna consisting of 10 articles; article 3 1.6 times as long as article 2; article 4 1.2 times as long as wide, 1.5 times as long as article 3; article 5 1.3 times as long as wide, 1.1 times as long as article 4; flagellum with 5 articles, terminal article terminating in 5 short simple setae, extending to middle of pereonite 1. Mandibular molar process ending in an acute incisor; mandibular palp article 3 with 7 simple setae. Maxillula simple with 4 terminal robust setae. Maxilla mesial lobe partly fused to lateral lobe; lateral lobe with 2 recurved robust setae; mesial lobe with 2 recurved robust setae. Maxilliped weakly segmented, with lamellar oostegite lobe, article 3 with 3 small robust setae.

Pereopod 1 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.3 times as long as wide; dactylus stout, 2.7 times as long as propodus, 3.8 times as long as wide. Pereopod 2 propodus 2.1 times as long as wide; dactylus 2.2 as long as propodus. Pereopod 6 basis 2.6 times as long as greatest width; ischium 0.5 times as long as basis; propodus 1.3 times as long as wide; dactylus 2.5 times as long as propodus. Pereopod 7 basis 3.2 times as long as greatest width; ischium 0.7 times as long as basis, without protrusions; merus proximal margin without bulbous protrusion; merus 1.1 times as long as wide, 1.6 times as long as ischium; carpus 1.5 times as long as wide, 0.5 times as long as ischium, without bulbous protrusion; propodus 2.6 times as long as wide, 0.8 times as long as ischium; dactylus slender, 1.8 times as long as propodus, 5.0 times as long as wide. Pereopod 7 with few setae on propodus, carpus, and merus.

Remarks

The description of A.haemuli from H.flavolineatum given above is in agreement with the original description in Bunkley Williams and Williams (1981). We supplement the original species diagnosis by now providing drawings and measurements of the antenna and antennula articles, additional pereopods, and pleopods.

Anilocrahaemuli from H.flavolineatum can be distinguished from all other Caribbean species based on the morphological and/or site attachment differences among species that were reported in Bunkley Williams and Williams (1981). Pereopods 2–4 do not swell on the outer margin of the dactyl, thereby excluding it from being Anilocraadudefdufi Bunkley Williams & Williams, 1981, A.holocanthi Bunkley Williams & Williams, 1981, A.chaetodontis, or A.partiti Bunkley Williams & Williams, 1981. In A.haemuli, the posterioventral angle of pereonite 6 is slightly produced thereby excluding it from being A.holocentri. The The endopod of the uropod of A.haemuli extends beyond the posterior end of the exopod, which is not the case in Anilocrachromis or A.partiti. Whereas the attachment site of A.haemuli is under the eye, A.holocentri and A.myripristis Bunkley Williams & Williams, 1981 attach between the eyes, and A.acanthuri Bunkley Williams & Williams, 1981 attaches under the pectoral fin.

Pereopod 1 basis 1.8 times as long as greatest width; ischium 0.23 times as long as basis; merus proximal margin without bulbous protrusion; carpus with straight proximal margin; propodus 1.9 times as long as wide; dactylus moderately slender, 1.8 times as long as propodus, 3.7 times as long as wide. Pereopod 2 propodus 1.7 as long as wide; dactylus 2.7 times as long as propodus, 4.9 times as long as wide. Pereopods gradually increasing in size towards posterior. Pereopod 6 basis 1.7 times as long as greatest width; ischium 0.7 times as long as basis; propodus 1.5 times as long as wide, dactylus 2.3 times as long as propodus, 3.8 times as long as wide. Pereopod 7 basis 3.0 times as long as greatest width; ischium 0.7 times as long as basis, without protrusions; merus proximal margin without bulbous protrusion, 2.0 times as long as wide, 0.7 times as long as ischium; carpus 1.5 times as long as wide, 0.6 times as long as ischium, without bulbous protrusion; propodus 3.2 times as long as wide, 0.8 times as long as ischium; dactylus moderately slender, 0.9 times as long as propodus, 3.5 times as long as wide. Pereopod 7 with many setae on propodus, carpus, and merus.

Etymology

This species is named in honor of Elizabeth R. Brill for her dedication to studying the ecology of A.haemuli, and for collecting many of the A.haemuli and A.brillae sp. n. specimens used in this study.

Distribution

Known from St. John and St. Thomas, USVI, Guana Island, BVI, and islands of Puerto Rico, Spanish Virgin Islands. Expected distribution throughout the Caribbean Sea, where fish of the Serranidae family inhabit.

Hosts

Known from Epinephelusguttatus (Linnaeus, 1758).

Remarks

Previously, A.brillae sp. n. was identified as A.haemuli. Compared to A.haemuli, the outer margins of the cephalon and pereonites 1–4 of A.brillae sp. n. form a more pronounced trapezoid shape and the remaining portion of the body is ovoid. A.brillae sp. n has more strongly narrowed pleonites than A.haemuli. Pleonites 1–3 of A.brillae sp. n. are wider than 4–5 and 4–5 are subequal, whereas the pleonites 1–2 of A.haemuli are wider than 3–5, and 3–5 are subequal. Pleonite 5 is more posteriorly rounded in A.brillae sp. n, but this is somewhat variable among individuals. Another more variable feature is A.brillae sp. n. has a more caudomedially pointed pleotelson than A.haemuli. Typically, the seventh pereopod of A.brillae sp. n. is proportionally larger, has more robust setae, and the setae are distributed more extensively over the articles when compared to A.haemuli. The antennula peduncle of A.brillae sp. n. is regularly observed as shorter and more robust than that of A.haemuli. With respect to attachment, both species infest the subocular region, and if infested by two parasites, one parasite typically attaches under each eye. Infestation by a third A.brillae sp. n. on a single host seems to occur with more frequency than tertiary infestation by A.haemuli on a single host. The third parasite is typically attached between the eyes on the head of the host, or adjacent to one of the other parasites (RLW, pers obs).

Anilocrabrillae sp. n. can be distinguished from all other Caribbean species except Anilocrahaemuli using the same morphological comparisons described between A.haemuli and other Anilocra spp. given in Bunkley Williams and Williams (1981). Additionally, the body of A.brillae sp. n. is not expanded and is more elongate compared to the bodies of A.holocanthi and A.chaetodontis.

Discussion

The results of this study provide the first reliable COI sequences for species of Anilocra, and confirm that A.haemuli from H.flavolineatum is morphologically and genetically different than the Anilocra specimens collected from E.guttatus, and are here described as A.brillae sp. n. Our morphological data suggest there are two different species given the number of differences consistently observed, and our molecular analyses demonstrate a 4% difference between A.haemuli and A.brillae sp. n. This difference is less than half of that observed between A.brillae sp. n. and A.chromis, which are more conspicuously morphologically different. Our supplemental analyses were conducted utilizing the available Anilocra sp. COI sequences on GenBank, and there was a high level of interspecific divergence of these sequences compared with our dataset. The large differences in interspecific divergence between the specimens of this study and those provided on GenBank may be explained by the fact that the GenBank specimens may have been misidentified or not identified at all, as no morphological identification was described in Ketmaier et al. (2007). Thus, further interspecific comparisons cannot be assessed at this time.

Anilocra spp. have been reported to influence the fitness (Adlard and Lester 2004, Fogelman et al. 2009) and behavior (Meadows and Meadows 2003, Welicky and Sikkel 2015, Welicky et al. in press) of their fish hosts, and Anilocrabrillae sp. n. infests E.guttatus, a grouper species that is currently recovering from previously intense fishing pressure (Nemeth et al. 2005). There is limited knowledge on the biotic stressors that influence E.guttatus population dynamics, and thus the effects of A.brillae sp. n. on E.guttatus should be examined as a potential stressor. Moreover, by studying this host-parasite interaction, further insight into variations in life histories of Anilocra spp. may be gained, if the life cycle of the parasite coincides with that of their host. The only complete description of an Anilocra spp. life cycle is of a species that infests an egg laying/guarding fish species (Adlard and Lester 1995), whereas many Anilocra spp. infest broadcast spawners. Interestingly, A.brillae sp. n. infests a fish species that undergoes an annual long distance migration to spawn in an aggregation (Nemeth 2011). Given that Anilocra spp. infection has been reported to influence host swimming performance in some fish (e.g., Binning et al. 2013), A.brillae sp. n. infection may indirectly influence the reproductive success of their hosts.

This study exemplifies that there is an incomplete but growing knowledge of cymothoid life histories, genetics, and morphology, and how these disciplines relate to host-parasite ecology. Continued efforts to conduct studies in these disciplines are necessary to better understand one of the least understood parasite families.

Acknowledgements

The financial assistance of the National Research Foundation (NRF) of South Africa supported this research and is hereby acknowledged (NRF project IFR2011040100022, NJ Smit, PI). Opinions expressed, and conclusions arrived at, are those of the authors and are not necessarily those of the NRF. Financial assistance for KA Hadfield and RL Welicky from the Claude Leon Foundation of South Africa for this research is acknowledged. A portion of the fieldwork reported herein was supported by the U.S. National Science Foundation (OCE-121615, PC Sikkel, PI) and Puerto Rico Sea Grant (project number R-31-1-14, PC Sikkel, PI). The Falconwood Corporation supported work at Guana Island. Drs. Bert and Lucy Williams are acknowledged for the specimens they collected during 1976–1977 and 1983 that were used in this study. This is the first in a series of publications emanating from the Williams’ parasitic isopods collection hosted by the NWU-Water Research Group. We thank C. Cook and E.C. Netherlands for their assistance with the molecular component of this study, and O. Kudlai and C. Baillie for their feedback on the preparation of this manuscript. We also thank the Sikkel lab, the staff of the Virgin Islands Environmental Resource Station, US National Parks Service (St. John), and Guana Island for logistic support, and E.R. Brill for field and collections assistance. This is contribution number 173 from the Center for Marine and Environmental Studies, University of the Virgin Islands and contribution number 179 from the NWU-Water Research Group.

Ellis JP (1981) Some type specimens of Isopoda (Flabellifera) in the British Museum (Natural History), and the isopods in the Linnaean Collection. Bulletin of the British Museum (Natural History) 40: 121–128.

Fogelman RM, Kuris AM, Grutter AS (2009) Parasitic castration of a vertebrate: effect of the cymothoid isopod, Anilocraapogonae, on the five-lined cardinalfish, Cheilodipterusquinquelineatus. International Journal for Parasitology. 39: 577–583. https://doi.org/10.1016/j.ijpara.2008.10.013

Hadfield KA, Bruce NL, Smit NJ (2013) Review of the fish-parasitic genus Cymothoa Fabricius,1783 (Isopoda, Cymothoidae, Crustacea) from the south-western Indian Ocean, including a new species from South Africa. Zootaxa 3640: 152–176. https://doi.org/10.11646/zootaxa.3640.2.2